Abstract. Tuber melanosporum (TM) is an edible fungus that exhibits antioxidant and anti-tumor activity via its unique bioactive metabolites. The present study analyzed the anti-fatigue effects of TM using a BALB/c mouse model. The anti-fatigue properties of TM were evaluated by assessing the endurance of mice by performing forced swimming, rotary rod and running tests. Following 2 weeks TM treatment, hepatic and muscular ATP, and glycogen levels were increased in mice subjected to 30 min swimming, compared with controls. Similarly, levels of serum lactic acid and lactic dehydrogenase were decreased in the same group, compared with the control. Additionally, TM treatment reduced reactive oxygen species and malondialdehyde levels, and increased superoxide dismutase and glutathione peroxidase levels in the muscle, liver and/or serum. The effect of TM on hormone levels was also investigated in the present study, as different efficacies of TM were observed in male and female mice. TM treatment increased serum levels of progesterone, estradiol and testosterone in female and male mice, whereas a decrease in serum luteinizing hormone levels was only observed in females. A decrease in serum follicle-stimulating hormone levels was identified in females, whereas an increase was observed in males. The current study demonstrated that the anti-fatigue effects of TM occur via the regulation of oxidative stress, energy metabolism and hormone levels.